The contribution of the cerebral microvasculature dysfunction to progressive ischemic injury after middle cerebral artery (MCA) occlusion requires investigation. Based on our preliminary data, we hypothesize that intravascular fibrin deposition contributes to progressive cerebral microvascular plasma perfusion deficit after embolic MCA occlusion, that increased levels of type 1 plasminogen activator (PAI-1) in the cerebral microvasculature contribute to stabilization of fibrin deposition and that intra-arterial administration of TNK-tPA, a thrombolytic agent resistant to PAI-1, to embolic ischemic rats accelerates recanalization of occluded cerebral vessels, improves cerebral microvascular perfusion, reduces infarct volume and improves neurological outcome. We will test our hypotheses in our newly developed model of focal cerebral embolic ischemia which mimics human stroke. We will measure temporal and spatial profiles of cerebral microvascular plasma perfusion deficits, fibrin deposition and parenchymal cell response to microvascular plasma perfusion deficit in ischemic brain by means of three dimensional quantitative laser scanning confocal microscopy (LSCM) in combination with immunofluorescent staining. We will determine whether changes in local levels of key fibrinolytic genes, t-PA, urokinase type plasminogen activator (u-PA), and PAI-1 contribute to fibrin deposition by means of reverse transcription (RT)-polymerase chain reaction (PCR), in situ hybridization, zymography and immunohistochemistry. We will determine whether intra-arterial administration of TNK-tPA accelerates recanalization of occluded vessels in ischemic brain by comparison with intravenous administration of TNK-tPA. Recanalization rate, tissue perfusion, microvascular plasma perfusion, ischemic lesion and neurological outcome will be assessed by means of MR angiography, MRI perfusion and diffusion weighted imaging, LSCM, histopathology and a battery of behavior tests. We expect that secondary microvascular perfusion deficit caused by intravascular fibrin deposition contributes to ischemic cell damage and intra-arterial administration of TNK-tPA accelerate recanalization of occlude vessels and reduces ischemic damage. Thus, data generated from this application will enhance our understanding of the mechanisms underlying secondary microvascular perfusion deficit resulting from MCA occlusion and provide new information on the optimum route for fast, and effective recanalization of occluded vessels with TNK-tPA.